PROJECT SUMMARY Neuronal function requires the reshaping and rewiring of neural connections through a fundamental process named synaptic plasticity. It is well known that the strength of neurotransmission is regulated by feedback signaling between the pre- and postsynaptic neuron, which leads to short- and/or long-term structural adaptations. However, little is known about how this regulation occurs. The current objective is to elucidate this missing mechanistic link by exploring emerging signaling roles of mitochondria, which are organelles that sustain the local energy demand of synaptic function and plasticity. Based on the rationale that neuronal activity sets the pace of aerobic energy conversion and of emission of chemically reactive oxygen species (ROS) from mitochondria, we hypothesize that controlled and localized mitochondrial emission of ROS regulates synaptic function and plasticity. Our preliminary data show that emission of mitochondrial ROS can be elicited specifically in presynaptic terminals in vivo. We will pioneer in the use of optogenetics for synchronized induction and measurement of ROS emission during synaptic function. Functional ROS signaling targets will be explored using super resolution fluorescence microscopy and mass spectrometry. Importantly, our collaborators and we established a method to elicit and study synaptic plasticity within just a few hours using optogenetics in vivo. Our results will impact therapies aimed improving neuronal communication in age-related neurodegeneration.